Rac and Cdc42 are members of the Rho family of small GTP binding proteins. These GTPases are involved signal transduction processes affecting morphogenesis and assembly of cytoskeletal structures involved in cell motility and metastasis. Recently, a family of serine/threonine protein kinases characterized by their binding to Cdc42 and Rac have been described. Based upon this characteristic, they have been named p21 -activated kinases (PAK). These constitute a new subfamily of kinases, characterized by high homology to the Saccharomyces cerevisiae Ste20 kinase.
A mammalian PAK (p65.sup.PAK) was identified by its specific binding to GTP-bound forms of Cdc42 and Rac1 (Manser et al, 1994). Previously, a gene for a second member of this family was isolated from a rat brain cDNA library based on its ability to interact specifically with the C-terminal portion of the Alzheimer .beta. amyloid precursor protein (a fragment comprising the C-terminal 100 amino acids of .beta.-APP). The gene encoding this protein appears to be specifically expressed in neuronal tissue. The sequence which was found to be closely related to that of p65.sup.PAK and to the yeast STE20 gene product (Ramer and Davis, 1993), was designated N-Pak (nervous system specific p.sup.21 activated kinase). The sequence of this N-Pak cDNA clone and corresponding amino acid sequence are disclosed in WO 94/05811, U.S. patent application Ser. Nos. 08/559,397, 08/144,555, and 07/938,184, the disclosures of which are incorporated herein by reference in their entirety.
In the last few years, numerous additional PAKs, characterized by the presence of a sequence conserved C-terminal kinase domain and upstream p21-binding and activation domains, have been reported. Thus, these kinases comprise a ubiquitous new family of serine/threonine kinases, referred to as the PAK/Ste20 family. Three mammalian isoforms have been described to date. In rodents, the 68-kDa .alpha.-PAK (Manser et al, 1994) and the 65 kDa .beta.-PAK (Manser et al, 1995; Bagrodia et al, 1995a) appear to be enriched in brain, while the 62 kDa .gamma.-isoform (Teo et al, 1995) is widely expressed in all tissues. Human homologues of the rat .alpha.-isoform, hPAK1 (Brown et al, 1996) and the .gamma.-isoforms hPAK2 (Knaus et al, 1995) and hPAK65 (Martin et al, 1995) have been described.
In addition to the numerous vertebrate species now shown to have PAK kinases, homologues have been described in insects such as Drosophila (Harden, et al. 1996) and several fungal species. Genes encoding Ste20 homologues have been isolated from the budding yeast S. cerevisiae (Ramer and Davis 1993; Cvrckova, et al, 1995), the fission yeast Schizosaccharomyces pombe (Ottilie et al, 1995; Marcus et al 1995) and the dimorphic human opportunistic pathogenic yeast Candida albicans (Leberer et al, 1996; Kohler and Fink, 1996)
In budding yeast, the Ste20 protein is a component of a MAPK signal transduction pathway leading to mating (Ramer and Davis, 1993). This pathway is activated by hormone stimulation of a trimeric G protein coupled mating receptor. Ste20 serves as a link between G.beta..gamma. and downstream mitogen-activated protein kinases of this well-studied signalling cascade and is therefore essential for activation of mating specific genes (Leberer et al, 1992; Wu et al, 1995). Recent evidence suggests an additional role for Cdc42 and Ste20 or Ste20 homologues in yeast morphogenesis (Cvrckova et al, 1995; Mosch, H-Uet al, 1996). Likewise, in fission yeast, disruption of the pak1 gene causes aberrant actin localization and reduces mating (Marcus et al, 1995; Ottilie et al, 1995).
Recent evidence suggests a role for mammalian Paks in signalling to p38 and JNK MAP kinases (Zhang et al, 1995; Bagrodia et al, 1995b; Brown et al, 1996). In addition, human PAK1 and PAK2 have been shown to be regulated by G-protein coupled receptors (Knaus et al, 1995). Therefore, there appears to be a common role for the PAK family members in signalling pathways which utilize Cdc42 and Rac1.
Given the homology of mammalian PAK family proteins to the yeast Ste20 protein, the ability of the mammalian protein to substitute for the yeast protein has been examined. Zhao et al, (1995) found that the rat p65PAK was unable to complement a S. cerevisiae ste20 mutant. They postulated that the mammalian enzyme was unable to phosphorylate the appropriate downstream target. Subsequently, the mouse gene m-PAK-3 was tested in a similar manner and found to give extremely weak suppression of the mating defect; mating occurred at a level that was 3-4% of a wild type strain (Bagrodia et al, 1995a). Brown et al (1996) reported that full length hPAK can restore mating of a ste20-null mutant to nearly normal wild type levels. This led these authors to suggest that the differences in amino acids (8 out of 545) between rat and human might be sufficient to account for this difference, and that one or more of these differences might define residues important for protein-protein interaction.
The establishment of this class of serine/threonine kinases as important intermediates in cell signal transduction has generated much interest in their activity and role in a variety of cellular processes such as differentiation, cytoskeletal assembly, motility and growth, particularly in light of the finding that the brain specific N-Pak protein binds specifically to the cytoplasmic domain of .beta.-APP. Heretofore, the expression of rat N-PAK in yeast has been unknown. We have discovered that N-PAK truncated to exclude the N-terminal region containing the Cdc42 binding domain can functionally substitute for the yeast Ste20 protein.